Engineering PD-L1/4-1BB-conjugated ATRA-loaded lipid nanoparticles for immune checkpoint inhibition and antitumor activity in melanoma

Işlek Köklü Z., Ucisik M. H., Taskan E., Sahin F., Ozer O.

JOURNAL OF DRUG DELIVERY SCIENCE AND TECHNOLOGY, cilt.126, ss.108784, 2026 (SCI-Expanded, Scopus)

Özet

Immune checkpoint blockade has reshaped cancer immunotherapy; however, therapeutic resistance and persistent immunosuppression within the tumor microenvironment continue to limit treatment success. All-trans retinoic acid (ATRA) possesses well-established immunomodulatory functions capable of reprogramming tumorassociated immune cells, yet its clinical translation is restricted by poor solubility and rapid degradation. In this study, we developed a dual-antibody, ATRA-loaded solid lipid nanoparticle (SLN) platform designed to integrate checkpoint inhibition and co-stimulation within a single nanoscale construct. Tripalmitin-based PEGylated SLNs were synthesized and conjugated with anti-PD-L1 and anti-4-1BB monoclonal antibodies using maleimide-thiol coupling to ensure surface functionalization. Comprehensive characterization by DLS, NTA, Bradford, and flow cytometry confirmed uniform spherical morphology (~179 nm, PDI < 0.25), a negative zeta potential (-43 mV), and high antibody conjugation efficiency (>85%). Biological evaluations in RAW264.7 macrophages and B16F10 melanoma cells-including MTS viability, cell-cycle analysis, Annexin V, qPCR, and PD-L1 profiling-demonstrated that ATRA-loaded SLNs induced dose-dependent cytotoxicity and apoptosis in melanoma cells with minimal macrophage toxicity. ATRA-loaded SLN formulation further enhanced apoptosis (up to 61%), triggered G2/M arrest, increased p53 and Bax expression, and markedly reduced PD-L1 levels. Splenocyte-melanoma co-culture assays demonstrated enhanced cytotoxicity following treatment with the antibody-conjugated ATRA-loaded SLN formulations. Overall, this rationally engineered nanoplatform provides a biocompatible and modular lipid-PEG antibody architecture capable of coordinated molecular targeting and immune activation. The system offers a promising foundation for next-generation immunotherapeutic nanocarriers applicable to treatment-resistant melanoma and other solid tumors.